Porcine reproductive and respiratory syndrome virus and methods of use

ABSTRACT

The present invention provides isolated European-like porcine reproductive and respiratory syndrome viruses, polynucleotides, and polypeptides. The present invention also provides methods for making antibodies to the viruses and polypeptides, methods for detecting porcine reproductive and respiratory syndrome viruses, immunogenic compositions, and methods for treating a porcine subject at risk of infection by, or displaying symptoms of, a porcine reproductive and respiratory syndrome virus infection.

CONTINUING APPLICATION DATA

This application claims the benefit of the following U.S. ProvisionalApplications: Ser. No. 60/181,041, filed Feb. 8, 2000; Ser. No.60/193,220, filed Mar. 30, 2000; Ser. No. 60/206,624, filed May 24.2000; Ser. No. 60/215,373, filed Jun. 29, 2000; and Ser. No. 60/260,041,filed Jan. 5, 2001, entitled PORCINE REPRODUCTIVE AND RESPIRATORYSYNDROME VIRUS AND METHOD OF DETECTION; each of which is incorporated byreference herein.

BACKGROUND

Porcine reproductive and respiratory syndrome virus (PRRSV) is a memberof the family Arteriviridae in the order Nidovirales (Cavanagh et al.,Virol., 176, 306–307 (1990)) that causes reproductive failure inbreeding swine and respiratory problems in young pigs (see Rossow, Vet.Pathol., 35, 1–20 (1998)). The syndrome was first recognized as a“mystery swine disease” in the United States in 1987 and was discoveredin Europe in 1990. A strain of PRRSV that is prevalent in Europe hasbeen isolated and is referred to as the Lelystad virus (Wensvoort etal., Vet. Q., 13, 121–130(1991)). A North American PRRSV, referred to asVR-2332, has been isolated (Collins et al., J. Vet. Diagn. Investig., 4,117–126 (1992)). The disease has also been referred to as Wabashsyndrome, mystery pig disease, porcine reproductive and respiratorysyndrome, swine plague, porcine epidemic abortion and respiratorysyndrome, blue abortion disease, blue ear disease, abortus blau, andseuchenhafter spatabort der schweine. The disease is characterized byreproductive failure in pregnant sows and respiratory problems in pigsof all ages. The disease has a significant negative impact on the swineindustry.

PRRSV is an enveloped positive single-stranded RNA virus. The 5′-cappedand 3′-polyadenylated RNA of the virus is polycistronic, containing (5′to 3′) two large replicase open reading frames (ORFs), 1a and 1b, andseveral smaller ORFs. In the infected cell, arteriviruses produce anested set of six to eight major coterminal subgenomic mRNAs (sgmRNAs)each thought to express only the relative 5′-terminal ORF. These sgmRNAshave a leader sequence derived from the 5′ end of the genome that isjoined at specific leader-body junction sites located downstream by anunclear discontinuous transcription mechanism (Lai, Adv. Exp. Med.Biol., 380, 463–471 (1995)). The sgmRNAs of PRRSV encode fourglycoproteins (GP2 to 5, encoded by sgmRNAs 2 to 5), an unglycosylatedmembrane protein (M, encoded by sgmRNA 6), and a nucleocapsid protein(N, encoded by sgmRNA 7). The European prototype strain of PRRSV,Lelystad, contains all six of these proteins in the virus particle, butonly the proteins encoded by ORFs 5 to 7 have conclusively beendemonstrated to be in the virion of North American isolates.

Nucleotide and amino acid sequence comparisons of the 3′-terminal ORFs 2to 7 have shown that there are significant differences between PRRSVstrains native to Europe and those found in North America (Kapur et al.,J. Gen. Virol., 77, 1271–1276 (1996), Murtaugh et al., Arch. Virol., 40,1451–1460 (1995)). Substantial variation also occurs among NorthAmerican PRRSV isolates. Genotypic comparison between strains VR-2332and Lelystad has revealed that ORF 1a of VR-2332 is vastly differentfrom that of Lelystad in both length and sequence, while ORF 1b isrelatively conserved between the two strains of PRRSV. The 5′ leadersequence of VR-2332 was 31 bases shorter than that of Lelystad anddiffered considerably in nucleotide sequence. Regional amino acidsequence comparisons also revealed that although the recognizedfunctional domains of the ORF 1a proteins were present in both strains,the proteins were not well conserved between these domains. Thus,although these two PRRSV strains cause similar diseases, they aredifferent in the genes encoding structural proteins.

PRRSV continues to cause significant economic losses throughout theworld. Vaccines are available, but they are based on one PRRSV strain,and there is evidence that PRRSV strains vary at the antigenic andgenetic levels. In addition, since the virus was identified in Europeand in the United States, new disease phenotypes have continued toemerge.

SUMMARY OF THE INVENTION

The present invention represents the identification of a novel porcinereproductive and respiratory syndrome virus (PRRSV). It is known to theart that there is a great deal of nucleotide sequence variation betweenEuropean PRRSV associated with European outbreaks of mystery swinedisease (MSD) and North American PRRSV associated with North Americanoutbreaks of MSD. As used herein the terms “European PRRSV” and“European strain” are used interchangeably and refer to strains of PRRSVthat are prevalent in Europe. An example of a “European PRRSV” that isknown to the art is the prototypic European strain, Lelystad, which isavailable from the Collection Nationale De Cultures De Microorganisms,Institut Pasteur, France, as deposit number I-1102 (see Wensvoort etal., U.S. Pat. No. 5,620,691). The nucleotide sequence of the Lelystadstrain is available at Genbank Accession Number NC_(—)002533. As usedherein the terms “North American PRRSV” and “North American strain” areused interchangeably and refer to strains of the PRRSV that areprevalent in North America. An example of a “North American PRRSV” thatis known to the art is the prototypic North American strain, VR-2332,which is available from the ATCC as deposit number VR-2332. Thenucleotide sequence of the VR-2332 strain is available at GenbankAccession Number U87392.

The PRRSV described herein has not been described before, and wasassociated with a North American outbreak of MSD, but unexpectedly andsurprisingly has a nucleotide sequence that has more similarity toEuropean PRRSV strains, than to North American PRRSV strains. As usedherein the phrase “European-like PRRSV” and “European-like strain” areused interchangeably and refer to PRRSV of the present invention. Thecharacteristics of European-like PRRSV are described herein.

The present invention provides an isolated virus deposited under ATCCAccession Number PTA-2194, and an isolated cell comprising the virus.Also provided by the invention is an isolated virus that includes an RNApolynucleotide that includes the RNA nucleotide sequence correspondingto SEQ ID NO: 1. The invention provides an isolated polynucleotide thatincludes the sequence SEQ ID NO: 1. The isolated polynucleotide can haveat least about 96% identity with a polynucleotide having the sequenceshown in SEQ ID NO: 1 using a GAP algorithm with default parameters,wherein the polynucleotide replicates in a cell.

Also provided is a vector that includes a polynucleotide that includesthe sequence shown in SEQ ID NO: 1, and a polypeptide that includes anamino acid sequence selected from the group consisting of SEQ IDNO:2–10. The invention provides polypeptides that have an amino acidsequence having at least about 95% identity to SEQ ID NO:2, at leastabout 99% identity to SEQ ID NO:3, at least about 98% identity to SEQ IDNO:4, at least about 94% identity to SEQ ID NO:5, at least about 95%identity to SEQ ID NO:6, at least about 91% identity to SEQ ID NO:7, atleast about 99% identity to SEQ ID NO:9, or at least about 99.5%identity to SEQ ID NO: 10.

The invention provides an antibody that specifically binds aEuropean-like porcine reproductive and respiratory syndrome virus(PRRSV), and a method of making an antibody. The method includesadministering to an animal a virus particle that includes an RNApolynucleotide that includes the RNA nucleotide sequence correspondingto SEQ ID NO: 1, or a polypeptide that includes an amino acid sequenceselected from the group consisting of SEQ ID NO:2–10, or apolynucleotide encoding the polypeptide. The particle, polypeptide, orpolynucleotide is administered in an amount effective to cause theproduction of an antibody specific for the virus particle. The antibodycan be a polyclonal antibody or a monoclonal antibody, and the methodcan further include isolating the antibody. Also provided is theantibody produced by the method.

Methods for detecting a PRRSV are also provided. A method includescontacting a virus particle, for instance from a biological sample, withan antibody of the present invention under conditions to form a complexwith a virus particle, and detecting the complex, wherein the presenceof the complex indicates the presence of a PRRSV. The method can also beused to detect PRRSV in a porcine subject. Also provided is a kit foruse in detecting PRRSV in a porcine subject. The kit includes theantibody of the invention and instructions for using the antibody.

Methods for detecting the presence of a European-like PRRSV are alsoprovided. The methods include contacting a viral polynucleotide with afirst primer and a second primer under conditions suitable to form adetectable amplification product. The first primer includes a nucleotidesequence that is complementary to nucleotides 2268 and 2269 of SEQ IDNO:1 or the complement thereof. The method further includes detecting anamplification product, wherein the detection indicates that the viralpolynucleotide is a European-like PRRSV. Examples of first primers thatcan be used include 5′ATCGGGAATGCTCAGTCCCCTT (SEQ ID NO:12), and5′-AAGGGGACTGAGCATTCCCG (SEQ ID NO:14). The method can also be used fordetecting the presence of a European-like PRRSV in a porcine subject,and includes contacting a biological sample of a porcine subject withthe first primer and the second primer. The biological sample preferablyincludes lung tissue.

Also provided by the invention is a kit for use in detecting PRRSV in aporcine subject. The kit includes the first primers and second primersof the invention suitable for use in amplification of a portion of aPRRSV and instructions for using the primer pair. Another kit providedby the invention is for use in detecting antibody to PRRSV in a porcinesubject. The kit includes the virus of the invention and instructionsfor using the virus.

Further provided by the invention is an immunogenic composition. Thecomposition includes an attenuated or inactivated PRRSV that includes apolynucleotide having at least about 96% identity with a polynucleotidehaving the sequence shown in SEQ ID NO: 1 using a GAP algorithm withdefault parameters. The immunogenic composition may include apolypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, an immunogenic analog thereof, an immunogenicfragment thereof, or a combination thereof.

Methods of treating a porcine subject at risk of infection with a PRRSVor displaying symptoms of a PRRSV infection are also provided. Themethods include administering to the animal an immunogenic compositionthat includes an attenuated or inactivated PRRSV that includes apolynucleotide having at least about 96% identity with comprising an RNApolynucleotide comprising the RNA nucleotide sequence corresponding toSEQ ID NO: 1 using a GAP algorithm with default parameters. Theimmunogenic composition is administered in an amount effective to causean immune response to the PRRSV. The immunogenic composition can includea polypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, an immunogenic analog thereof, an immunogenicfragment thereof, or a combination thereof. Alternatively, the porcinesubject can be administered a neutralizing antibody in an amounteffective to treat the porcine subject.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. DNA nucleotide sequence of a portion of the positive strand ofthe genome of the European-like strain (SEQ ID NO: 1). The RNA sequencethat corresponds to SEQ ID NO: 1 and is present in a viral particle hasuracil (U) nucleotides instead of the thymidine (T) residues. Rows 1, 2,and 3 under the nucleotide sequence represent the three differentreading frames. The predicted amino acid sequences encoded by theEuropean-like strain are depicted for some predicted open readingframes, including: SEQ ID NO:2 (ORF1 a), SEQ ID NO:3 (ORF1b), SEQ IDNO:4 (ORF2), SEQ ID NO:5 (ORF3), SEQ ID NO:6 (OFR4), SEQ ID NO:7 (ORF5),SEQ ID NO:8, SEQ ID NO:9 (ORF6), and SEQ ID NO: 10 (ORF7).

FIG. 2. DNA nucleotide sequence of a portion of the positive strand ofthe genome of the European-like strain (nucleotides 1,830 to 2,618 ofSEQ ID NO: 1) compared to a portion of the DNA nucleotide sequence ofthe prototypic European strain Lelystad (SEQ ID NO 11, which correspondsto nucleotides 1,981 to 2,820 of Genbank Accession Number NC_(—)002533).In SEQ ID NO: 11, the upper case nucleotides signify alignednon-identical nucleotides; lower case nucleotides signify unalignednucleotides; dashes signify aligned identical nucleotides; and dotssignify a gap.

DETAILED DESCRIPTION OF THE INVENTION

Polynucleotides

The present invention is based on the the identification of a novelporcine reproductive and respiratory syndrome virus (PRRSV), anenveloped positive single-stranded RNA virus. Accordingly, the presentinvention provides isolated polynucleotides. Preferably, an isolatedpolynucleotide can replicate in a cell. Preferably, an isolatedpolynucleotide of the present invention is no greater than about 15.3kilobases. Whether an isolated polynucleotide can replicate in a cellcan be determined by inserting the polynucleotide into an expressionvector, producing an infectious RNA, introducing the infectious RNA to acells, and evaluating if the infectious RNA causes the cell to producevirus particles. These methods are described in greater detail herein. Apreferred example of a polynucleotide of the present invention is SEQ IDNO: 1 (FIG. 1). This polypeptide is a portion of a polynucleotideobtained from a European-like PRRSV. Preferably, the European-like PRRSVis one having the strain designation MND99-35186, and deposited with theAmerican Type Culture Collection, 10801 University Blvd., Manassas, Va.,20110-2209, USA, on Jul. 7, 2000 (granted ATCC Accession NumberPTA-2194).

The deposit was made under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. It is expected that the complete nucleotide sequence of thePRRSV disclosed in SEQ ID NO: 1 will include additional nucleotides atthe 5′ end of the polynucleotide. Specifically, it is expected thatabout 100 to about 200, preferably about 150, additional nucleotides canbe present at the 5′ end of SEQ ID NO: 1 when the nucleotide sequence ofthe entire PRRSV represented by SEQ ID NO: 1 is determined. It should benoted that while SEQ ID NO: 1 is a DNA sequence, the present inventioncontemplates the corresponding RNA sequence, and RNA and DNA complementsthereof, as well.

As used herein, an “isolated” substance is one that has been removedfrom its natural environment, produced using recombinant techniques, orchemically or enzymatically synthesized. For instance, a polypeptide,polynucleotide, or virus particle of this invention can be isolated.Preferably, a polypeptide, polynucleotide, or virus particle of thisinvention is purified, i.e., essentially free from any other type ofpolypeptide, polynucleotide, or virus particle and associated cellularproducts or other impurities. As used herein, the term “polynucleotide”refers to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxynucleotides, and includes both double- andsingle-stranded DNA and RNA. Unless otherwise noted, a polynucleotideincludes the complement thereof. The nucleotide sequence of thecomplement of a polynucleotide can be easily determined by a person ofskill in the art. A polynucleotide may include nucleotide sequenceshaving different functions, including for instance coding sequences, andnon-coding sequences such as regulatory sequences and/or non-translatedregions. A polynucleotide can be obtained directly from a naturalsource, or can be prepared with the aid of recombinant, enzymatic, orchemical techniques. A polynucleotide can be linear or circular intopology. A polynucleotide can be, for example, a portion of a vector,such as an expression or cloning vector, or a fragment.

“Polypeptide” as used herein refers to a polymer of amino acids and doesnot refer to a specific length of a polymer of amino acids. Thus, forexample, the terms peptide, oligopeptide, protein, and enzyme areincluded within the definition of polypeptide. This term also includespost-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like.

The terms “coding region” and “coding sequence” are used interchangeablyand refer to a polynucleotide region that encodes a polypeptide and,when placed under the control of appropriate regulatory sequences,expresses the encoded polypeptide. The boundaries of a coding region aregenerally determined by a translation start codon at its 5′ end and atranslation stop codon at its 3′ end. A regulatory sequence is apolynucleotide sequence that regulates expression of a coding region towhich it is operably linked. Nonlimiting examples of regulatorysequences include promoters, transcription initiation sites, translationstart sites, translation stop sites, and terminators. “Operably linked”refers to a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Aregulatory sequence is “operably linked” to a coding region when it isjoined in such a way that expression of the coding region is achievedunder conditions compatible with the regulatory sequence.

“Complement” and “complementary” refer to the ability of two singlestranded polynucleotides to base pair, i.e., hybridize, with each other,where an adenine of one polynucleotide will base pair to a thymine of asecond polynucleotide and a cytosine of one polynucleotide will basepair to a guanine of a second polynucleotide. Two polynucleotides arecomplementary to each other when a nucleotide sequence in onepolynucleotide can base pair with a nucleotide sequence in a secondpolynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. Theterms complement and complementary also encompass two polynucleotideswhere one polynucleotide contains at least one nucleotide that will notbase pair to at least one nucleotide present on a second polynucleotideunder the hybridization conditions described below. For instance thethird nucleotide of each of the two polynucleotides 5′-ATTGC and5′-GCTAT will not base pair, but these two polynucleotides arecomplementary as defined herein.

The present invention also provides isolated polynucleotides thatcorrespond to the coding regions present in SEQ ID NO:1. These codingregions are shown in Table 1.

TABLE 1 Coding regions of SEQ ID NO: 1 Nucleotides of SEQ ID NO: 1corresponding Polypeptide encoded by the SEQ ID NO of the to the codingregion. coding region. polypeptide.    71 to 7,210 ORF1a SEQ ID NO: 2 7,207 to 11,583 ORF1b SEQ ID NO: 3 11,594 to 12,343 ORF2 SEQ ID NO: 412,202 to 12,994 ORF3 SEQ ID NO: 5 12,744 to 13,295 ORF4 SEQ ID NO: 613,292 to 13,897 ORF5 SEQ ID NO: 7 13,449 to 13,775 not applicable SEQID NO: 8 13,885 to 14,406 ORF6 SEQ ID NO: 9 14,396 to 14,782 ORF7 SEQ IDNO: 10

The present invention also includes polynucleotides having structuralsimilarity to SEQ ID NO:1 or to a coding region present in SEQ ID NO:1.The similarity is referred to as “percent identity” and is determined byaligning the residues of the two polynucleotides (i.e., the nucleotidesequence of a candidate polynucleotide and the nucleotide sequence ofSEQ ID NO:1 or a coding region of SEQ ID NO: 1) to optimize the numberof identical nucleotides along the lengths of their sequences; gaps ineither or both sequences are permitted in making the alignment in orderto optimize the number of shared nucleotides, although the nucleotidesin each sequence must nonetheless remain in their proper order. Acandidate polynucleotide is the polynucleotide that has the nucleotidesequence being compared to SEQ ID NO:1 or to a coding region present inSEQ ID NO: 1 (e.g., nucleotides 71 to 7,210 of SEQ ID NO:1). A candidatepolynucleotide can be isolated from an animal, preferably a pig infectedwith PRRSV, or can be produced using recombinant techniques, orchemically or enzymatically synthesized. Preferably, two nucleotidesequences are compared using the GAP program of the GCG WisconsinPackage (Genetics Computer Group, Madison, Wis.) version 10.0 (updateJanuary 1999). The GAP program uses the algorithm of Needleman andWunsch (J. Mol. Biol., 48, 443–453 (1970)) to find the alignment of twocomplete sequences that maximizes the number of matches and minimizesthe number of gaps. Preferably, the default values for all GAP searchparameters are used, including scoring matrix=NewsgapDNA.cmp, gapweight=50, length weight=3, average match=10, average mismatch=0. In thecomparison of two nucleotide sequences using the GAP search algorithm,structural similarity is referred to as “percent identity.” Preferably,a polynucleotide includes a nucleotide sequence having a structuralsimilarity with a coding region of SEQ ID NO:1 of at least about 96%,more preferably at least about 98%, most preferably at least about 99%identity.

Another isolated polynucleotide provided by the invention is an RNApolynucleotide to which an oligonucleotide having the sequenceAGAGCGGGAACAGAATCCTTCCCACCTTTAGCGGTACGCTTG (SEQ ID NO:18) hybridizes.Preferably, the RNA polynucleotide replicates in cells to form virusparticles. Such an RNA is referred to as an infectious RNA. Theproduction and testing of infectious RNAs are described in greaterdetail below. Preferably, hybridization conditions include denaturingabout 1 μg total RNA with glyoxyl and electrophoresing through a 2%agarose gel, transferring to a nylon membrane (MagnaGraph, MSI,Westboro, Mass.), and crosslinking to the membrane by ultraviolet light.Preferably, the oligonucleotide is 3-end radiolabeled, for instance with[α³²P]dATP (Amersham Life Science, Arlington Heights, Ill.) and terminaldeoxynucleotide transferase (TdT) (Promega Corporation, Madison, Wis.).Preferably, hybridization conditions include incubation of the membranecontaining the crosslinked RNA with the labeled oligonucleotide in ahybridization solution, for instance QuikHyb (Stratagene, La Jolla,Calif.) at 68° C. for 16 hours. The membrane is then washed 3 times in asolution containing 0.9 M sodium chloride/0.09 M sodium citrate/pH 7.0(6×SSC) and 0.5% sodium dodecyl sulfate (SDS) at 78° C., and thenexposed to autoradiography film (NEN Life Science Products, Boston,Mass.) or a phosphoimaging screen (Molecular Dynamics, Inc., Sunnyvale,Calif.). It is expected that under these conditions, the oligonucleotidewill not hybridize to the European PRRSV Lelystad or to the NorthAmerican PRRSV VR-2332.

Preferably, a polynucleotide of the present invention includes adeletion when compared to the nucleotide sequence of European strainLelystad, which is available at Genbank Accession Number NC_(—)002533.When the nucleotide sequence of SEQ ID NO:1 and Genbank Accession NumberNC_(—)002533 are compared, nucleotides 2,419 to 2,470 of GenbankAccession Number NC_(—)002533 are not present in SEQ ID NO: 1.Nucleotides 2268 and 2269 of SEQ ID NO: 1 are immediately 5′ (upstream)and 3′ (downstream) of this deletion. Thus, those polynucleotides of thepresent invention that include nucleotides 2268 and 2269 of SEQ ID NO:1include this deletion. The presence of this deletion is useful indistinguishing between a polynucleotide of the present invention andsome PRRSV clinical isolates (described in greater detail herein).

The isolated polynucleotides of the present invention can be obtainedfrom a virus particle. As used herein, the terms “virus particle” and“viral particle” are used interchangeably and refer to a PRRSV particle.A virus particle includes an RNA polynucleotide that will reproduce in acell, for instance a cell in a pig and/or a cultured primary (i.e.,freshly isolated) porcine alveolar macrophage, under the appropriateconditions. A virus particle also includes an envelope that surroundsthe polynucleotide. A virus particle is typically obtained from a pigpresenting symptoms of mystery swine disease (MSD), including abortion,anorexia, fever, lethargy, pneumonia, red/blue discoloration of ears,labored breathing (dyspnea), and increased respiratory rate (tachypnea).While not intending to be limiting, a virus particle can be obtainedfrom such a pig by the removal of tissue, preferably lung tissue,followed by microscopic examination of the tissue for thickened alveolarseptae caused by the presence of macrophages, degenerating cells, anddebris in alveolar spaces. These characteristics indicate the presenceof an infection by a PRRSV. The lung or other porcine tissue is thenhomogenized with a pharmaceutically acceptable aqueous solution (such asphysiological saline, Ringers solution, Hank's Balanced Salt Solution,Minimum Essential Medium, and the like) such that the tissue includesabout 10 percent weight/volume amount of the homogenate. The virus canbe isolated by low speed centrifugation as described in Example 1 toform a homogenate. Alternatively, the virus can be isolated by passingthe homogenate through filters with pore diameters in the 0.05 to 10micron range, preferably through a series of 0.45, 0.2 and 0.1 micronfilters, to produce a homogenate containing the PRRSV. As a result, thehomogenate contains viral particles having a size no greater than about1.0 micron, preferably no greater than about 0.2 to 0.1 micron. Othertissues, including fetal tissue, may also be used to recover virus.Typically, such a virus particle is then grown in vivo (i.e., within thebody of a subject) or in cell culture (i.e., in vitro) to produce morevirus particles. This process of infecting an animal or a cell inculture, allowing the virus to reproduce, and then harvesting the newlyproduced virus is referred to herein as passaging the virus. Optionally,the virus is purified.

The homogenate described above can be passaged in cell culture byinoculation into a series of cultured cells. Cultured cells can bemammalian organ cells such as kidney, liver, heart and brain, lung,spleen, testicle, turbinate, white and red blood cells and lymph nodecells, as well as insect and avian embryo preparations. Preferably, thecell is a primary porcine alveolar macrophage. Preferably, primaryporcine alveolar macrophages are isolated from at least two pigs, andthe primary porcine alveolar macrophages from each pig are not combined.It has been observed that there is some variability in the ability ofthe virus of the present invention to replicate in primary porcinealveolar macrophage, and the use of primary porcine alveolar macrophagesfrom more than one pig significantly increases the ability to passagethe virus in the macrophages. Culture media suitable for these cellpreparations include those supporting mammalian cell growth such asserum (for instance, fetal calf serum or swine serum) and agar, bloodinfusion agar, brain-heart infusion glucose broth and agar and the like.After inoculating cultured cells with homogenate and growing theculture, individual clumps of cultured cells can be harvested andreintroduced into sterile culture medium with cells. Alternatively andpreferably, supernatants from cultured cells are subjected to low speedcentrifugation and used to inoculate sterile culture medium containingcells.

Whether an isolated, preferably purified, virus particle obtained inthis way is able to cause MSD can be determined by inoculation of 3 to 4week old pigs as described in Example 1, or by the methods of Terpstraet al., (Vet. Q., 13, 131–136 (1991)), and Collins et al., (U.S. Pat.No. 5,846,805). These methods experimentally test if the viral particlereproduces late term abortion and reproductive failure in pregnant sowsor clinical signs and microscopic lesions in gnotobiotic piglets similarto field outbreaks. Pigs experimentally inoculated in this manner canalso be used for in vivo passage of the virus by collecting tissue andprocessing for the isolation of virus as described in Example 1.

After isolation, preferably purification, of the virus particle, thepolynucleotide in the particle can be isolated by, for instance,treating the particle to remove the envelope. Methods for removing theenvelope are known in the art and include, for instance, solubilizingwith phenol:chloroform or guanidunium. Optionally, the polynucleotide ispurified using methods known to the art, including, for instance,precipitating the polynucleotide.

The polynucleotides of the present invention can be present in a vector.A vector is a replicating polynucleotide, such as a plasmid, phage,cosmid, or artificial chromosome to which another polynucleotide (e.g.,a polynucleotide of the present invention) may be attached so as tobring about the replication of the attached polynucleotide. When apolynucleotide of the present invention is in a vector thepolynucleotide is DNA. When present in a vector, a polynucleotide of theinvention can be referred to as a “recombinant polynucleotide.”Construction of vectors containing a polynucleotide of the inventionemploys standard ligation techniques known in the art. See, e.g.,Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold SpringHarbor Laboratory Press (1989) or Ausubel, R. M., ed. Current Protocolsin Molecular Biology (1994).

A vector can provide for further cloning (amplification of thepolynucleotide), i.e., a cloning vector, or for expression of thepolypeptide encoded by a coding region present in the polynucleotide,i.e., an expression vector. Selection of a vector depends upon a varietyof desired characteristics in the resulting construct, such as aselection marker, vector replication rate, and the like. Suitable hostcells for cloning or expressing the vectors herein are prokaryote oreukaryotic cells, and suitable vectors for cloning and/or expression inprokaryote and/or eukaryote cells are known to the art. Typically, whenthe vector is used to clone a polynucleotide, the host cell is aprokaryote. Suitable prokaryotes include eubacteria, such asgram-negative or gram-positive organisms. Preferably, E. coli is used.Host cells suitable for expression of the polypeptides of the inventionare described in greater detail below.

The polynucleotide used to transform the host cell optionally includesone or more marker sequences, which typically encode a molecule thatinactivates or otherwise detects or is detected by a compound in thegrowth medium. For example, the inclusion of a marker sequence canrender the transformed cell resistant to an antibiotic, or it can confercompound-specific metabolism on the transformed cell. Examples of amarker sequence are sequences that confer resistance to kanamycin,ampicillin, chloramphenicol, tetracycline, neomycin, and formulations ofphleomycin D1 including, for example, the formulation available underthe trade-name ZEOCIN (Invitrogen).

An expression vector optionally includes regulatory sequences operablylinked to the coding sequence. The invention is not limited by the useof any particular promoter, and a wide variety are known. Promoters actas regulatory signals that bind RNA polymerase in a cell to initiatetranscription of a downstream (3′ direction) coding sequence. Thepromoter used in the invention can be a constitutive or an induciblepromoter. It can be, but need not be, heterologous with respect to thehost cell. Examples of promoters for use in vectors present inprokaryotic cells include lac, lacUV5, tac, trc, T7, SP6 and ara.

Promoter sequences are known for eukaryotes. Most eukaryotic codingsequences have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is the CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be asignal for addition of the poly A tail to the 3′ end of the codingsequence. All these sequences are suitably inserted into eukaryoticexpression vectors.

Transcription of a coding sequence encoding a polypeptide of the presentinvention in mammalian host cells can be controlled, for example, bypromoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, andHepatitis-B virus.

Transcription of a coding sequence encoding a polypeptide of the presentinvention by eukaryotes can be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually having about 10 to 300 bp, that act on a promoter to increaseits transcription. Enhancers are relatively orientation- andposition-independent, having been found 5′ and 3′ to coding sequences,within an intron as well as within the coding sequence itself. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, alpha-fetoprotein, and insulin). Enhancers from eukaryotic cellviruses are also known and include the SV40 enhancer on the late side ofthe replication origin, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the coding sequence encoding a polypeptide of thepresent invention, but is preferably located at a site 5′ of thepromoter.

An expression vector can optionally include a ribosome binding site (aShine Dalgarno site for prokaryotic systems or a Kozak site foreukaryotic systems) and a start site (e.g., the codon ATG) to initiatetranslation of the transcribed message to produce the enzyme. It canalso include a termination sequence to end translation. A terminationsequence is typically a codon for which there exists no correspondingaminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotideused to transform the host cell can optionally further include atranscription termination sequence. The rrnB terminators, which is astretch of DNA that contains two terminators, T1 and T2, is an oftenused terminator that is incorporated into bacterial expression systems.Transcription termination sequences in vectors for eukaryotic cellstypically include a polyadenylation signal 3′ of the coding sequence.

Suitable host cells for expression vector that includes a polynucleotideencoding a polypeptide of the invention can be derived frommulticellular organisms. Such host cells are capable of processing andglycosylation activities. Vertebrate or invertebrate culture can beused. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodoptera frugiperda,Aedes aegypti, Aedes albopictus, Drosophila melanogaster, Trichoplusiani, and Bombyx mori are known to the art.

Vertebrate cells can also be used as hosts. Examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (CAS-7,ATCC CRL-1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, Graham et al., J. Gen. Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO); CHO-K1 (ATCC CCL-61); CHO-D; mouse sertolicells (TM4); monkey kidney cells (CV1, ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (WI38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL 51); TRI cells; MRC 5 cells; FS4 cells; ahuman hepatoma line (Hep G2), MARC-145 (Kim et al., Arch. Virol., 133,477–483 (1993)), and MA-104 (ATCC CRL-2378).

An expression vector of the present invention can be used to determineif a polynucleotide of the present invention replicates in a cell. Apolynucleotide of the invention replicates in a cell if the cell cultureshows signs of cytopathic effect (CPE) as described in Example 2, and/orif virus particles can be isolated from the cells. The polynucleotidepresent in the expression vector can be transcribed in vitro (i.e., cellfree) to produce RNA transcripts. The RNA transcripts can be introducedinto cultured cells, and incubated under conditions suitable forreplication of a PRRSV. RNA transcripts that replicate will use thecellular machinery (including, for instance, ribosomes, and tRNAs) toreplicate. The culture can be assayed as described in Example 2 for CPE.The presence of CPE indicates the virus is able to replicate in a cell.Optionally, the virus particles produced by the cells can be isolated.This type of expression vector is often referred to in the art as aninfectious cDNA clone, and the RNA produced by the expression vector isreferred to as an infectious RNA. Methods for cloning the European PRRSVLelystad and inserting it into a vector are known to the art (Meulenberget al., J. Virol., 72, 380–387 (1998)), and it is expected that thepolynucleotides of the present invention can be used in this way toproduce infectious RNAs. Moreover, the method of Meulenberg et al. canbe used to make viral particles. Accordingly, a person of skill in theart can provide a polynucleotide of the present invention, for instanceSEQ ID NO:1, introduce the polynucleotide into an expression vector, andproduce infectious RNAs that could be introduced to cells to result inthe production of virus particles. The cells transfected with aninfectious RNA can be, for instance, BHK-21 cells, CL-2621 cells, MA-104cells, MARC-145 cells, or primary porcine alveolar macrophages,preferably primary porcine alveolar macrophages. Methods for efficientlytransfecting cells include the use of calcium chloride, and commerciallyavailable products known under the trade names LIPOFECTIN andLIPOFECTAMINE. Methods for efficiently transfecting primary porcinealveolar macrophages are known to the art (Groot Bramel-Verheige et al.,Virol., 278, 380–389 (2000)).

Polypeptides

The present invention is also directed to polypeptides, preferablyisolated polypeptides, encoded by polynucleotides of the presentinvention. Preferably, a polypeptide of the present invention hasimmunogenic activity. “Immunogenic activity” refers to an amino acidsequence which elicits an immunological response in a subject. Animmunological response to a polypeptide is the development in a subjectof a cellular and/or antibody-mediated immune response to thepolypeptide. Usually, an immunological response includes but is notlimited to one or more of the following effects: the production ofantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells (see, for example, de Antonio et al., Vet. Immunol.Immunopathol., 61, 265–277 (1998), and Kwang et al., Res. Vet. Sci., 67,199–201 (1999)) directed specifically to an epitope or epitopes of thepolypeptide fragment. As used herein, an antibody that can “specificallybind” or is “specific for” a virus particle and/or a polypeptide is anantibody that interacts only with an epitope of the antigen that inducedthe synthesis of the antibody, or interacts with a structurally relatedepitope. An antibody that “specifically binds” a European-like PRRSV isan antibody that does not specifically bind a European PRRSV, preferablya European PRRSV having deposit number I-1102, or a North AmericanPRRSV, preferably a North American PRRSV having deposit number VR-2332.As used herein, the term “complex” refers to the combination of anantibody and a virus particle and/or a polypeptide that results when anantibody specifically binds to a virus particle and/or a polypeptide.

Preferred examples of polypeptides of the invention are SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10 (see Table 1). The present inventionfurther includes polypeptides having structural similarity with thepolypeptides of the present invention. The structural similarity isreferred to as percent identity and is generally determined by aligningthe residues of the two amino acid sequences (i.e., a candidate aminoacid sequence and the amino acid sequence of one of SEQ ID NOs:2–10) tooptimize the number of identical amino acids along the lengths of theirsequences; gaps in either or both sequences are permitted in making thealignment in order to optimize the number of identical amino acids,although the amino acids in each sequence must nonetheless remain intheir proper order. A candidate amino acid sequence is the amino acidsequence being compared to an amino acid sequence present in a preferredpolypeptide of the present invention. Preferably, two amino acidsequences are compared using the GAP program of the GCG WisconsinPackage (Genetics Computer Group, Madison, Wis.) version 10.0 (updateJanuary 1999). The GAP program uses the algorithm of Needleman andWunsch (J. Mol. Biol., 48, 443–453 (1970) to find the alignment of twocomplete sequences that maximizes the number of matches and minimizesthe number of gaps. Preferably, the default values for all GAP searchparameters are used, including scoring matrix=BLOSUM62.cmp, gapweight=8, length weight=2, average match=2.912, and averagemismatch=−2.003. In the comparison of two amino acid sequences using theGAP search algorithm, structural similarity is referred to as “percentidentity.” Preferably, a polypeptide includes an amino acid sequencehaving a structural similarity with SEQ ID NO:2 of at least about 95%,more preferably at least about 97%, most preferably at least about 99%identity. Preferably, a polypeptide includes an amino acid sequencehaving a structural similarity with SEQ ID NO:3 of at least about 99%identity. Preferably, a polypeptide includes an amino acid sequencehaving a structural similarity with SEQ ID NO:4 of at least about 98%,more preferably at least about 99% identity. Preferably, a polypeptideincludes an amino acid sequence having a structural similarity with SEQID NO:5 of at least about 94%, more preferably at least about 96%, mostpreferably at least about 99% identity. Preferably, a polypeptideincludes an amino acid sequence having a structural similarity with SEQID NO:6 of at least about 95%, more preferably at least about 97%, mostpreferably at least about 99% identity. Preferably, a polypeptideincludes an amino acid sequence having a structural similarity with SEQID NO:7 of, in increasing order of preference, at least about 91%, atleast about 93%, at least about 95%, at least about 97%, most preferablyat least about 99% identity. Preferably, a polypeptide includes an aminoacid sequence having a structural similarity with SEQ ID NO:9 of atleast about 99% identity. Preferably, a polypeptide includes an aminoacid sequence having a structural similarity with SEQ ID NO: 10 of atleast about 99.5% identity.

The present invention further includes polypeptide analogs andpolypeptide fragments, preferably immunogenic polypeptide analogs andimmunogenic polypeptide fragments. Preferably, a polypeptide fragment isat least about 8, more preferably at least about 12, most preferably atleast about 20 amino acids in length. Immunogenic analogs ofpolypeptides of the present invention include polypeptides having aminoacid substitutions that do not eliminate the ability of the polypeptideto elicit an immunological response. Substitutes for an amino acid maybe selected from other members of the class to which the amino acidbelongs. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, aspartate, and glutamate. Thepositively charged (basic) amino acids include arginine, lysine, andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of preferred conservative substitutionsinclude Lys for Arg and vice versa to maintain a positive charge; Glufor Asp and vice versa to maintain a negative charge; Ser for Thr sothat a free —OH is maintained; and Gln for Asn to maintain a free NH₂.

Immunogenic analogs, as that term is used herein, also include modifiedpolypeptides. Modifications of polypeptides of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Immunogenicfragments of a polypeptide include a portion of the polypeptidecontaining deletions or additions of one or more contiguous ornoncontiguous amino acids such that the resulting polypeptide isimmunogenic.

The polypeptides of the present invention can be obtained from, forinstance, a biological sample from a porcine subject infected with aEuropean-like PRRSV that encodes the polypeptide. Preferably, theEuropean-like PRRSV is one that includes SEQ ID NO: 1, more preferablyit is the European-like PRRSV having ATCC number PTA-2194. Thepolypeptide can be obtained from cultured cells, preferably primaryporcine alveolar macrophages, that have, for instance, been infectedwith a European-like PRRSV that encodes the polypeptide or contain arecombinant polynucleotide, preferably a polynucleotide of theinvention, that encodes a polypeptide of the invention. Alternatively,the polypeptide can be obtained from a prokaryotic cell or a eukaryoticcell that contains an expression vector that includes a polynucleotideencoding a polypeptide of the invention. The polypeptides of the presentinvention can also be obtained by chemical synthesis.

Viruses

The present invention includes isolated European-like virus particles.The European-like virus particles of the present invention include apolynucleotide having structural similarity to SEQ ID NO: 1, preferablyat least about 96%, more preferably at least about 98%, most preferablyat least about 99% identity to SEQ ID NO: 1. A preferred example of avirus particle is one that includes SEQ ID NO: 1. More preferably, thevirus particle is the virus having ATCC Accession Number PTA-2194. Avirus particle of the present invention include an envelope, and can,when added to a cultured cell, can replicate to result in the productionof more viral particles.

As discussed above, a virus particle of the present invention can beobtained from a pig presenting symptoms of MSD. A virus particle can begrown by passage in vivo or in cell culture. Passage in vivo includesinoculating a pig, for instance as described in example 1. Passage incell culture includes exposing cultured cells to the virus particle andincubating the cells under conditions suitable for the virus toreproduce and produce more virus particles. Preferably, the culturedcells are not an immortalized or transformed cell line (i.e., the cellsare not able to divide indefinitely). Preferably, primary porcinealveolar macrophages are used for passage in cell culture. The use ofprimary porcine alveolar macrophages is described in Example 2. A virusparticle can also be obtained from cells transfected with an infectiousRNA as described herein.

A virus of the present invention can be inactivated, i.e., renderedincapable of reproducing in vivo and/or in cell culture. Methods ofinactivation are known to the art and include, for instance, treatmentof a virus of the invention with a standard chemical inactivating agentsuch as an aldehyde reagent including formalin, acetaldehyde and thelike; reactive acidic alcohols including cresol, phenol and the like;acids such as benzoic acid, benzene sulfonic acid and the like; lactonessuch as beta propiolactone and caprolactone; and activated lactams,carbodiimides and carbonyl diheteroaromatic compounds such as carbonyldiimidazole. Irradiation such as with ultraviolet and gamma irradiationcan also be used to inactivate the virus.

Also included in the present invention are attenuated European-likePRRSV (i.e., viruses having reduced ability to cause the symptoms of MSDin pigs), and methods of making an attenuated European-like PRRSV.Methods of producing an attenuated virus are known to the art.Typically, a virus of the present invention is passaged, i.e., used toinfect a cell in culture, allowed to reproduce, and then harvested. Thisprocess is repeated until the virulence of the virus in pigs isdecreased. For instance, the virus can be passaged 10 times in cellculture, and then the virulence of the virus measured. If virulence hasnot decreased, the virus that was not injected into the animal ispassaged an additional 10 times in cell culture. This process isrepeated until virulence is decreased. In general, virulence is measuredby inoculation of pigs with virus, and evaluating the presence ofclinical symptoms and/or LD₅₀ (see, for instance, Example 1, Halbur etal., J. Vet. Diagn. Invest., 8, 11–20 (1996), and Halbur et al., Vet.Pathol., 32, 200–204 (1995), and Park et al., Am. J. Vet. Res., 57,320–323 (1996)). Preferably, virulence is decreased so the attenuatedvirus does not cause the death of animals, and preferably does not causeclinical symptoms of the disease.

Typically, a cell culture useful for producing an attenuated virus ofthe present invention includes cells of non-porcine mammal origin.Examples of non-porcine mammal cell cultures include, for instance, thecell line MA-104 (ATCC CRL-2378), the cell line MARC-145 (Kim et al.,Arch. Virol., 133, 477–483 (1993)), and the cell line CL-2621 (Baustiaet al., J. Vet. Diagn. Invest., 5, 163–165 (1993)). Preferably, a mixedcell culture is used for producing an attenuated virus of the presentinvention. In a mixed cell culture there are at least two types of cellspresent. Preferably, a mixed cell culture includes an immortalized ortransformed cell line and a primary cell culture. A mixed cell cultureis particularly useful when a virus reproduces slowly, or not at all, inan immortalized or transformed cell line. Preferred examples of animmortalized or transformed cell line for use in a mixed cell cultureinclude, for example, the cell line MARC-145 (Kim et al., Arch. Virol.,133, 477–483 (1993)), and the cell line MA-104 (ATCC CRL-2378).Preferably, primary cell cultures for use in a mixed cell culture areporcine in origin. A preferred example of a primary cell culture for usein a mixed cell culture is primary porcine alveolar macrophages.

Methods of Use

The virus particles, polynucleotides, polypeptides, and immunogenicanalogs and immunogenic fragments thereof of the present invention canbe used to produce antibodies. Laboratory methods for producing,characterizing, and optionally isolating polyclonal and monoclonalantibodies are known in the art (see, for instance, Harlow E. et al.Antibodies: A laboratory manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor (1988). For instance, a virus of the presentinvention can be administered to an animal, preferably a mammal, in anamount effective to cause the production of antibody specific for theadministered virus. Polypeptides of the present invention andimmunogenic analogs and immunogenic fragments thereof can also beadministered to an animal, preferably a mammal, to produce antibodies.Optionally, a virus particle or a polypeptide is mixed with an adjuvant,for instance Freund's incomplete adjuvant, to stimulate the productionof antibodies upon administration. Preferably, the antibody is amonoclonal antibody.

Preferably, an antibody produced using a virus of the present invention,or a polypeptide or immunogenic analog or immunogenic fragment thereof,is a neutralizing antibody. A neutralizing antibody is one that preventsa virus of the present invention from reproducing in cell culture,preferably in primary porcine macrophages.

Optionally and preferably, antibody produced using a virus particle ofthe present invention, a polypeptide or polynucleotide of the presentinvention, or immunogenic analogs and immunogenic fragments thereof donot specifically bind to the European PRRSV deposited as I-1102 with theCollection Nationale De Cultures De Microorganisms, Institut Pasteur,France, or to the North American PRRSV deposited as VR-2332 with theATCC. Whether an antibody of the present invention specifically binds toeither or both of those viruses can be determined using methods known tothe art.

The present invention provides methods for detecting a PRRSV, preferablya virus of the present invention. These methods are useful in, forinstance, detecting a PRRSV in an animal, detecting a PRRSV in a cellculture, or diagnosing a disease caused by a PRRSV. Preferably, suchdiagnostic systems are in kit form. Kits are described in greater detailbelow. In some aspects of the invention, detecting a PRRSV includesdetecting antibodies that specifically bind to a virus of the presentinvention, a polypeptide of the present invention, and/or an immunogenicanalog or immunogenic fragment thereof. The method includes providing abiological sample, preferably a liquid homogenate of a tissue sample,from a porcine subject. The subject can be suspected of harboring thePRRSV, or can be a member of a herd that is being screened for thepresence of the PRRSV. Antibody is added to the biological sample andincubated under conditions to form a complex with a PRRSV in thebiological sample. Preferably the antibody is produced using a virusparticle of the present invention, a polypeptide or polynucleotide ofthe present invention, or an immunogenic analog or immunogenic fragmentthereof. Preferably, the antibody does not specifically bind a EuropeanPRRSV or a North American PRRSV. The complex is then detected, and thepresence of the complex indicates the presence of a PRRSV in thebiological sample. The detection of antibodies is known in the art andcan include, for instance, immunofluorescence and peroxidase. Typicalformats in which antibodies of the present invention can be usedinclude, for instance, enzyme linked immunosorbent assay (ELISA);radioimmunoassay (RIA), immunofluorescent assay (IFA), and westernimmunoassay.

As used herein, a “biological sample” refers to a sample of tissue orfluid obtained from a subject, including but not limited to, forexample, lung or respiratory tract. A “biological sample” also includessamples of cell culture constituents including but not limited to thecells and media resulting from the growth of cells and tissues inculture medium. The cells can be infected with PRRSV or can contain avector that includes a polypeptide of the present invention, andpreferably includes a coding region encoding a polypeptide of thepresent invention.

Other methods for detecting a PRRSV, preferably a European-like PRRSV,include the amplification of a polynucleotide, preferably by thepolymerase chain reaction (PCR). The polynucleotide can be one that is,for instance, present in a biological sample from a porcine subject thatis suspected of harboring the PRRSV, or a member of a herd that is beingscreened for the presence of the PRRSV. The polynucleotide can beobtained from an isolated, preferably purified, virus particle. When thepolynucleotide is obtained from a virus particle, the polynucleotide isconverted from an RNA polynucleotide to a DNA polynucleotide by reversetranscription (see, for instance, Example 3). In some aspects of thepresent invention, the methods to detect a European-like PRRSV anddistinguish it from a European PRRSV and a North American PRRSV exploitthe presence of a deletion present in European-like PRRSV. This deletionis described above in the section labeled “Polynucleotides.”

In one aspect of detecting a PRRSV by amplification of a polynucleotide,the invention is directed to detecting a virus of the present inventionunder conditions where European PRRSV and North American PRRSV are notdetected. The method includes contacting a viral polynucleotide that issuspected of being a European-like PRRSV with a primer pair andincubating under conditions to form a detectable amplifiedpolynucleotide. As used herein, a “primer pair” refers to two singlestranded polynucleotides that can be used together to amplify a regionof a polynucleotide, preferably by a polymerase chain reaction (PCR).The polynucleotide that results from amplifying a region of apolynucleotide is referred to as an “amplification product” or an“amplified polynucleotide.” The phrase “under conditions suitable toform a detectable amplification product” refers to the reactionsconditions that result in an amplification product. For instance, in thecase of a PCR, the conditions suitable to form a detectableamplification product include the appropriate temperatures, ions, andenzyme.

One of the primers of the primer pair is complementary to a portion ofthe viral polynucleotide that corresponds to nucleotides 2268 and 2269of SEQ ID NO: 1, or the complement thereof. The use of such a primerresults in the production of an amplified polynucleotide when there is adeletion. In contrast, the use of such a primer with a European PRRSVwill not result in the production of an amplified polynucleotide becausethere are about 51 nucleotides present between the nucleotides thatcorrespond to nucleotides 2268 and 2269 of SEQ ID NO: 1, or thecomplement thereof. For instance, the use of such a primer pair will notresult in an amplified polynucleotide when the viral polynucleotide isfrom the Lelystad PRRSV. An example of a primer pair that can be used inthis method includes forward primer 5′ATCGGGAATGCTCAGTCCCCTT (SEQ IDNO:12), which corresponds to nucleotides 2,255 to 2,276 of SEQ ID NO:1and reverse primer Euro2714 5′-GCGCATAAGACAGATCCA (SEQ ID NO:13), whichis expected to result in an amplified polynucleotide of about 467nucleotides. Another primer pair is reverse primer:5′-AAGGGGACTGAGCATTCCCG (SEQ ID NO:14), which corresponds to thecomplement of nucleotides 2,257 to 2,276 of SEQ ID NO:1 and forwardprimer Euro20/5′-CAGAAGGGTTCGAGGAAG (SEQ ID NO:15), which is expected toresult in an amplified polynucleotide of about 170 nucleotides.

In another aspect of detecting a PRRSV by amplification of apolynucleotide, the invention is directed to detecting a virus of thepresent invention under conditions where both European-like PRRSV andEuropean PRRSV are detected, and the molecular weights of the amplifiedpolynucleotides vary. The primer pair used in this aspect produces anamplified polynucleotide that includes the region of the deletion, i.e.,nucleotides 2268 and 2269 of SEQ ID NO: 1, and the correspondingnucleotides of a European PRRSV. When both a European-like PRRSV andEuropean PRRSV are amplified, and the resulting amplifiedpolynucleotides are compared, the amplified polynucleotide that resultsfrom the European-like PRRSV will have a molecular weight that is about51 nucleotides less than an amplified polynucleotide from a EuropeanPRRSV. Methods of determining the approximate molecular weight of anamplified polynucleotide are known in the art, and include, forinstance, resolving the polynucleotide on an acrylamide or agarose gel.

An example of a primer pair that can be used in this method includesforward primer Euro1671/5′-GCCTGTCCTAACGCCAAGTAC (SEQ ID NO: 16) andreverse primer/Euro3165-rc: 5′-CATGTCCACCCTATCCCACAT (SEQ ID NO:17),which results in an amplified polynucleotide in a European-like PRRSV ofabout 1,494 nucleotides, and an amplified polynucleotide in a EuropeanPRRSV of about 1,544 nucleotides. Other primer pairs include forwardprimer Euro20/5′-CAGAAGGGTTCGAGGAAG (SEQ ID NO: 15) and reverseprimer/Euro3207 5′-GCTTGGAACTGCGAGG (SEQ ID NO: 19) (expected size ofamplified polynucleotide from a European-like PRRSV: about 910nucleotides); forward primer Euro20/5′-CAGAAGGGTTCGAGGAAG (SEQ ID NO:15)and reverse primer/Euro2714 5′-GCGCATAAGACAGATCCA (SEQ ID NO: 13)(expected size of amplified polynucleotide from a European-like PRRSV:about 616 nucleotides). When these primers are used to amplify aEuropean PRRSV, the size of the amplified polynucleotide is expected tobe about 51 nucleotides greater.

In another aspect of detecting a PRRSV by amplification of apolynucleotide, the invention is directed to detecting a virus of thepresent invention under conditions where European PRRSV is detected andEuropean-like PRRSV are not detected. In this aspect of the invention,at least one of the primers of a primer pair is complementary a portionof nucleotides 2,419 to 2,470 of the prototype European PRRSV, Lelystad(Genbank Accession number NC_(—)002533), of the complement thereof. Anexample of a primer pair that can be used in this method includesforward primer Euro1/: 5′-TGAAGGTGCTCTGGTCT (SEQ ID NO:20) and reverseprimer/Euro2: 5′-AAATTCCCGCCTACC (SEQ ID NO:21), which results in anamplified polynucleotide from a European PRRSV of about 51 nucleotides.

The present invention also provides a kit for detecting a virus of thepresent invention, and a kit for detecting a polypeptide of the presentinvention or an immunogenic analog or immunogenic fragment thereof. Thekit includes an antibody that specifically binds a virus of the presentinvention, a polypeptide of the present invention or immunogenic analogor immunogenic fragment thereof (when detecting the presence of thevirus) or a primer pair as described herein (when amplifying apolynucleotide) in a suitable packaging material in an amount sufficientfor at least one assay. Preferably, the antibody does not specificallybind to the European PRRSV deposited as I-1102 with the CollectionNationale De Cultures De Microorganisms, Institut Pasteur, France, or tothe North American PRRSV deposited as VR-2332 with the ATCC. The presentinvention also provides a kit for detecting antibody to a virus of thepresent invention, a polypeptide of the present invention or animmunogenic analog or immunogenic fragment thereof. When detectingantibody to the virus, polypeptide, or immunogenic analog or immunogenicfragment thereof the kit includes a virus of the present invention, apolypeptide of the present invention or immunogenic analog orimmunogenic fragment thereof. Optionally, other reagents such as buffersand solutions needed to practice the invention are also included.Instructions for use of the packaged virus or polypeptide or primer pairare also typically included.

As used herein, the term “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the polypeptide or primer pair can be usedfor detecting a virus of the present invention. In addition, thepackaging material contains instructions indicating how the materialswithin the kit are employed to detect a virus of the present invention.As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic, paper, foil, and the like, capable of holdingwithin fixed limits a virus or a primer pair. Thus, for example, apackage can be a glass vial used to contain milligram quantities of aprimer pair, or it can be a microtiter plate well to which microgramquantities of a virus have been affixed. “Instructions for use”typically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like.

The present invention is also directed to vaccines and methods oftreatment. Treatment can be prophylactic or, alternatively, can beinitiated after the development of MSD in a porcine subject. A vaccinecan include, for instance, an immunogenic composition or a neutralizingantibody. The term “vaccine” refers to a composition that, uponadministration to a subject, will provide protection against a virus ofthe present invention. When the vaccine includes an immunogeniccomposition, administration to the subject will also produce animmunological response to a polypeptide and result in immunity. Animmunogenic composition of the present invention can include anattenuated or inactivated virus of the present invention, and/or one ormore polypeptides of the present invention or immunogenic analogs orimmunogenic fragments thereof, and/or a polynucleotide.

As used herein, the term “immunogenic composition” refers to acomposition or preparation administered in an amount effective to resultin some therapeutic benefit or effect so as to result in an immuneresponse that inhibits or prevents MSD in a subject, or so as to resultin the production of antibodies to a PRRSV of the present invention.Both local and systemic administration is contemplated. Systemicadministration is preferred.

A polynucleotide used in a vaccine of the invention is preferably onethat includes a nucleotide sequence encoding a polypeptide on thepresent invention, or an immunogenic analog or immunogenic fragmentthereof. The polynucleotide can include DNA, RNA, or a combinationthereof. The polynucleotide can be supplied as part of a vector or as a“naked” polynucleotide. General methods for construction, production andadministration of polynucleotide vaccines are known in the art, e.g. F.Vogel et al., Clin. Microbiol. Rev. 8:406–410 (1995); WO 93/02556;Felgner et al., U.S. Pat. No. 5,580,859, Pardoll et al., Immunity 3:165(1995); Stevenson et al., Immunol. Rev. 145:211 (1995); Molling, J. Mol.Med. 75:242 (1997); Donnelly et al., Ann. N.Y. Acad. Sci. 772:40 (1995);Yang et al., Mol. Med. Today 2:476 (1996); and Abdallah et al., Biol.Cell 85:1 (1995)). A nucleic acid molecule can be generated by meansstandard in the art, such as by recombinant techniques, or by enzymaticor chemical synthesis.

Preferably, administration of a polynucleotide that is part of a vaccineincludes the introduction of an expression vector that includes thepolynucleotide. There are numerous plasmids known to those of ordinaryskill in the art useful for the production of polynucleotide vaccineplasmids, including, for instance, the plasmid pVAX1 as the vector (InVitrogen Corporation, Carlsbad, Calif.). In addition, the vectorconstruct can contain immunostimulatory sequences that stimulate theanimal's immune system. Examples of immunostimulatory sequences include,for instance, sequences with CpG motifs, two 5′ purines, an unmethylatedCpG dinucleotide, or two 3′ pyrimidines (see, for instance, Lowie etal., DNA Vaccines Methods and Protocols, Humana Press, Totowa, N.J.(2000)). Other possible additions to the polynucleotide vaccineconstructs include nucleotide sequences encoding cytokines, such asgranulocyte macrophage colony stimulating factor (GM-CSF) orinterleukin-12 (IL-12). The cytokines can be used in variouscombinations to fine-tune the response of the animal's immune system,including both antibody and cytotoxic T lymphocyte responses, to bringout the specific level of response needed to produce an immune response.Alternatively, the vaccine vector can be a viral vector, including anadenovirus vector, and adenovirus associated vector, or a retroviralvector.

Immunogenic carriers can be used to enhance the immunogenicity of avaccine that includes an immunogenic composition. Such carriers includebut are not limited to other polypeptides, polysaccharides, liposomes,and bacterial cells and membranes. Polypeptide carriers may be joined tothe attenuated or inactivated virus of the present invention, and/or apolypeptide of the present invention or an immunogenic analog ofimmunogenic fragment thereof to form fusion polypeptides by recombinantor synthetic means or by chemical coupling. Useful carriers and means ofcoupling such carriers to polypeptide antigens are known in the art.

The vaccine preferably includes a pharmaceutical carrier that iscompatible with a porcine subject. The vaccine may be delivered orally,parenterally, intranasally or intravenously. Factors bearing on thevaccine dosage include, for example, the age, weight, and level ofmaternal antibody of the infected pig. The vaccine doses should beapplied over about 14 to 28 days to ensure that the pig has developed animmunity to the MSD infection.

The vaccine of the present invention can be administered in a variety ofdifferent dosage forms. An aqueous medium containing the vaccine may bedesiccated and combined with pharmaceutically acceptable inertexcipients and buffering agents such as lactose, starch, calciumcarbonate, sodium citrate formed into tablets, capsules and the like.These combinations may also be formed into a powder or suspended in anaqueous solution such that these powders and/or solutions can be addedto animal feed or to the animals' drinking water. These compositions canbe suitably sweetened or flavored by various known agents to promote theuptake of the vaccine orally by the pig.

For purposes of parenteral administration, the composition can becombined with pharmaceutically acceptable carrier(s) well known in theart such as saline solution, water, propylene glycol, etc. In this form,the vaccine can be parenterally, intranasally, and orally applied bywell-known methods known in the art of veterinary medicine. The vaccinecan also be administered intravenously by syringe. In this form, thevaccine is combined with pharmaceutically acceptable aqueous carrier(s)such as a saline solution. The parenteral and intravenous formulationsof the composition may also include emulsifying and/or suspending agentsas well, together with pharmaceutically acceptable diluent to controlthe delivery and the dose amount of the composition.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Detection of Virus in Infected Pigs

This example describes the isolation of a new PRRSV from infected pigs.The PRRSV was given the designation MND99-35186 and is referred to inthese examples as European-like.

Methods

Three live, 3 to 4-day-old pigs from a herd with a clinical history oflate-gestation abortions and weak-born pigs were submitted to theMinnesota Veterinary Diagnostic Laboratory (St. Paul, Minn.). The pigswere euthanized by intravenous over dose of barbiturate and necropsied(autopsied).

Portions of lung, lymph nodes, brain, spleen, kidney, tonsil and heartwere collected and pooled as one sample. The sample was treated for theisolation of porcine reproductive and respiratory syndrome virus(PRRSV), pseudorabies virus (PRV), and swine influenza virus (SIV).

For isolation of PRRSV, samples were inoculated on MARC-145 cells andprimary porcine alveolar macrophages. Before inoculation, the sampleswere treated as described by Rossow et al. (Vet. Pathol., 32, 361–373(1995)). Briefly, Hank's Balanced Salt Solution was added to the tissuesample or sera to make an approximately 10% (vol/vol) suspension, andthen homogenized. The homogenate was centrifuged a 4,133×g for 20minutes, and the supernatant removed and saved. The pelleted materialwas discarded. The conditions for inoculating MARC-145 cells and primaryporcine alveolar macrophages are described below. In addition, serumfrom each pig was pooled together into one sample and then used toinoculate MARC-145 cells and primary porcine alveolar macrophages.

Portions of lung, lymph nodes, stomach, brain, liver, kidney, tonsil,heart and ileum (a section of the small intestine) were preserved in 10%formalin buffered with a mixture of dibasic sodium phosphate andmonobasic sodium phosphate to yield pH 7.0. The tissues were incubatedin the formalin for at least 12 hours before subsequent use in assays.Portions of each tissue were paraffin embedded.

Formalin fixed tissues were stained by hematoxylin and eosin (H and E)staining.

Formalin fixed tissues were also assayed for PRRSV testing byimmunohistochemical technique by the method of Christopher-Hennings etal., (Vet. Pathol., 35, 260–267 (1998)). Briefly, formalin fixed,paraffin embedded lung was sectioned at approximately 4 microns andsections were applied to glass slides. Tissue sections were covered withthe monoclonal antibody SDOW-17 and incubated in a humidified chamber.SDOW-17 is an anti-PRRSV monoclonal antibody that recognizes an epitopepresent on the Lelystad PRRSV and on the VR-23332 PRRSV. Antibodybinding to PRRSV in the lung was identified using a modified avidinbiotin complex method (Hsu et al., J. Histochem. Cytochem., 29, 577–580(1981)).

Lung sections were rapidly frozen in isopentane at −30° C. The frozensections were examined for PRRSV by direct fluorescent antibodytechnique using the monoclonal antibody SDOW-17. Direct FA examinationof tissue was done by the method of Rossow et al. (Vet. Pathol., 32,361–373 (1995)). Briefly, tissues were frozen, sectioned atapproximately 5 microns and transferred to glass slides. Tissue sectionswere covered with fluoroscene-conjugated SDOW-17, an anti-PRRSVmonoclonal antibody (obtained from E. Nelson, South Dakota StateUniversity, South Dakota). The tissue sections were then incubated in ahumidified chamber for about 1 hour, and excess unbound antibody removedby washing in phosphate buffered saline. The presence of antibodybinding to PRRSV in the lung was visualized with a fluorescentmicroscope.

Brain, lung, and liver samples were cultured on blood agar plates todetect the presence of aerobic bacteria.

Pooled tissues were also examined for leptospira using the method ofSmith et al. (Cornell Vet., 57, 517–526 (1967)).

Results

PRRSV was identified in lung sections from each pig byimmunohistochemical and direct fluorescent antibody.

Light microscopic tissue lesions (H and E slides) were compatible withPRRSV infection. Histopathology of the lungs showed diffuse septalthickening by macropahges. Some alveoli contained necrotic cell debris.Lymph nodes were characterized by germinal centers filled withblast-lymphocytes and small foci of necrosis. The muscular layer in onestomach was characterized by lymphoplasmacytic perineuritis andperivasculitis. Brain, liver, kidney, tonsil, heart, and ileum did nothave lesions.

Tests for PRV and SIV were negative and no bacterial pathogens wereidentified in tissues from the infected pigs.

PRRSV was isolated from pooled tissue homogenate and pooled seracultured in the alveolar macrophages. However, few cells were infectedwith PRRSV. No PRRSV was isolated from either sample cultured inMARC-145 cells.

Because this PRRSV grew poorly in the alveolar macrophages, 3 to4-week-old pigs from a documented PRRSV-free farm were inoculated(intramuscularly and intranasally) with approximately 1 ml of the virusobtained from the supernatant from the infected alveolar macrophages.The virus was diluted by adding about 9 mls of Hanks Balanced SaltSolution to about 1 ml of virus-containing supernatant. Clinically, theexperimentally infected pigs exhibited the same symptoms of mild,transient signs of lethargy within about 1–2 days that are also seenafter infection of a pig with Lelystad virus or VR-2332. Each infectedpig seroconverted to the PRRSV infection. Seroconversion was measured bythe IDEXX Elisa test (HerdChek-PRRSV, IDEXX Laboratories Inc. Westbrook,Me.). Seroconversion was also measured by indirect fluorescent antibodytest using Lelystad infected cells and the method of Yoon et al. (J.Vet. Diagn. Invest., 4, 144–147 (1992)). The European-like PRRSV wasre-isolated from infected pig tissues and serum, cultured in porcinealveolar macrophages, and identified in tissues by immunohistochemistry.

Example 2 Infection of Porcine Alveolar Macrophages with PRRSV

Porcine alveolar macrophages were isolated by collection fromPRRSV-negative pigs less than 6-weeks-old. Pigs were euthanized, andtrachea and lungs removed and airways lavaged with sterile phosphatebuffered saline. The phosphate buffered saline was made by combining 8.5grams NaCl, 1.1 grams disodium phosphate, and 0.32 gram sodiummonophosphate in 10 liters distilled water. The Porcine alveolarmacrophages were concentrated by centrifugation, confirmed negative forPRRSV by isolation and examination using direct fluorescent antibody asdescribed in Example 1, and used immediately or stored in liquidnitrogen at a concentration of 10⁶ cells/ml. Frozen alveolar macrophageschould be used within 6 months.

Freshly harvested porcine alveolar macrophages (about 10⁷) or frozencells (10⁶ cells) were plated on a 1×48 well plate or a 1×75 cm flask,and allowed to adhere for 4 hours to overnight in about 10 to 25 mlRPMI-1640 complete medium. The cells cannot be allowed to incubate formore than one day before virus is added. RPMI-1640 complete medium ismade by combining 500 ml RPMI-1640 medium containing 300 mg/literL-glutamine, 25 mM HEPES[N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)] (Catalog#10-041-CV, Mediatech Inc., Herndon. Va.), 40 ml heat-inactivated FetalBovine Serum (Cat #12133-78P; JRH Bioscience, Inc., Lenexa, Kans.), 1gram neomycin sulfate (Gibco Life Technologies, Rockville, Md.), 40 mlHanks Balanced Salt Solution (HBSS) (Gibco Life Technologies, #14180053,Rockville, Md.) containing neomycin sulfate at a final concentration of150 μg/ml medium), 66 ml HBSS containing penicillin G potassium salt(Sigma #P7794, St. Louis, Mo.), streptomycin sulfate (Sigma #S9137), andamphotericin B solubilized (Sigma A-9528) at a final concentration of2500 U penicillin G, 0.45 mg streptomycin sulfate/ml medium, and 120μg/ml amphotericin B/ml of medium.

The medium was removed, and the cells were washed in once ins HBSS, andthe cells were infected with 1 ml of virus in 1 ml RPMI incompletemedium (same as RPMI complete but without FBS added). The virus titercan vary, and when the virus is from tissue isolated from aPRRSV-infected pig, is unknown. HBSS was 500 mls HBSS supplemented with5 mls of 100× neomycin (10 mg/ml) and 5 mls of 100× pennecillin (10,000U ml), streptomycin (10 mg/ml), and fungizone (25 μg/ml).

The plate was gently rotated for 1 hour at room temperature. Nine mls ofRPMI complete medium were added and the infected cells were incubated at37° C. The cell were observed daily until 60–80% cytopathic effect (CPE)was seen (2–3 days). CPE appears as fragmented, vacuolated, malformed,shrunken cells.

Virus was isolated by removing the medium and centrifuging at about4,000×g for 10 minutes to remove cellular debris, and leave the virus inthe supernatant.

The presence of PRRSV in the primary porcine alveolar macrophages wasconfirmed by staining with the SDOW-17 monoclonal antibody as describedby Nelson et al. (J. Clin. Microbiol., 34, 3184–3189 (1993)).

Example 3 Sequence Analysis of Virus

1. PRRSV RNA Extraction:

Viral RNA was extracted from macrophage culture supernatants usingQIAamp Viral RNA Mini Spin Kit (QIAgen, Inc., Valencia, Calif.).Briefly, 280 μl cell culture fluid was added to 1120 μl BufferAVL/Carrier RNA, pulse vortexed for 15 seconds, incubated at roomtemperature for 10 minutes, and centrifuged briefly. After this step,1120 μl 100% ethanol was added to the reaction, pulse vortexed for 15seconds, and centrifuged briefly. The reaction was then applied to aQIAamp spin column (in a 2 ml collection tube) in 630 μl aliquots (4×)and centrifuged at 6000×g (800 rpm) for 1 minute, discardingflow-through each time. When all sample was applied to the filter, theQIAamp spin column was placed into a clean 2 ml collection tube, andagain centrifuged. Buffer AW1 (500 μl) was added to the filtercontaining viral RNA, and this reaction was centrifuged at 6000×g (8000rpm) for 1 minute. Buffer AW2 (500 μl) was added to the spin column andthe column was centrifuged at full speed (20,000×g; 14,000 rpm) for 3minutes. The collection tube was discarded, and the QIAamp spin columnwas place in a clean 1.5 ml centrifigue tube. Buffer AVE (60 μl) wasadded to the spin column, the column incubated at room temperature for 1minutes, and then centrifuged at 6000×g (8000 rpm) for 1 minutes.

2. RT-PCR: Two methods were used to reverse transcribe the viral RNA andobtain DNA for use in DNA sequence analysis. In general, the primerswere selected to hybridize to an appropriate portion of SEQ ID NO: 1 andamplify a DNA fragment that was then used in DNA sequencing reactions.a. Qiagen OneStep RT-PCR:Five microliters of extracted RNA were added to a reaction mixcontaining 1× Qiagen RT-PCR Buffer; 400 nM of each DATP, dCTP, dGTP anddTTP; 0.08 units/reaction RNase inhibitor (20 U/μl, Perkin Elmer, BostonMass.); 1/25 volume of Qiagen Enzyme mix; 300 nM each forward andreverse primer; to make a total reaction volume of 25 ml. Thethermocycling conditions consisted of: 1 cycle 50° C. for 30 minutes; 1cycle of 95° C. for 15 minutes; 35 cycles of 57° C. for 30 seconds, 72°C. for 45 seconds, 94° C. for 45 seconds; 1 cycle of 57° C. for 30seconds; 1 cycle of 72° C. for 10 minutes and a 4° C. hold.b. RT and PCR-2 Step Reactionb1. Reverse Transcription: Random primed cDNA was generated in thefollowing way: 2 μl of 50 μM random hexamers were added to 6 μl of RNAextract. This was heated to 70° C. for 5 minutes and quickly chilled onice. Then 32 μl of a master mix containing 5 mM MgCl₂ (Perkin Elmer), 1×Perkin Elmer Buffer II (50 mM KCl, 10 mM Tris-HCl, (pH 8.3 at roomtemperature)), 1 mM dNTPs (Perkin Elmer), 1 U/μl RNase Inhibitor (20U/μl, Perkin Elmer), and 25 U/μl MuLV RT (murine leukemia virus reversetranscriptase; Perkin Elmer). The thermocyling conditions consisted of:1 cycle of 22° C. for 10 minutes; 1 cycle of 42° C. for 15 minutes; 1cycle of 95° C. for 10 minutes, 1 cycle of 5° C. for 5 minutes and holdat 4° C.b2. PCR: Reactions tube containing 40 μl of 5 mM MgCl₂ (Perkin Elmer),1× Perkin Elmer Buffer II, 300 nM forward primer, 300 nM reverse primer,and 0.25 U/μl Amplitaq Polymerase (Perkin Elmer) was added to 10 μl cDNAobtained from the reverse transcription (paragraph b1, above).Alternatively, to amplify longer section of random primed cDNA, ExpandLong Template PCR Kit (Boehringer Mannheim, Indianapolis, Ind.) wasused. The thermocycling conditions consisted of: 1 cycle of 93° C. for 4minutes; 35 cycles of 57° C. for 30 seconds, 72° C. for 45 seconds, 93°C. for 45 seconds; 1 cycle of 57° C. for 30 seconds; 1 cycle of 72° C.for 10 minutes and a 4° C. hold. (Annealing temperature would varyaccording to the primer pair utilyzed to amplify cDNA).3. The Results of Each PCR were Evaluated and Prepared for DNASequencing.Sequence analysis was performed by the Advanced Genetic Analysis Center(AGAC) (University of Minnesota, Minneapolis, Minn.). using an ABI Model377 DNA Sequencer.I. Evaluation of PCR Reactions on an Agarose Gel.

One gram of agarose was added to 100 ml of 1× TAE buffer. This wasmicrowaved for 2 minutes, and 4 μl of 10 mg/ml EtBr was added to every100 ml agarose. The gel was cast and allowed to solidify for about 15–30minutes. Four μl of PCR product were mixed with 1 μl loading dye andadded to the gel, which was run at 140 volts for 1 hour or 75 volts for2 hours.

II. Purification of PCR Product with Qiagen Qiaquick PCR PurificationKit

For each sample to be purified, a column was placed into a collectiontube. One hundred μl PB buffer were added to the 20 μl PCR reaction leftin PCR tube, and mixed thoroughly. All of the PCR product/PB buffer mixwas added to the column, and the column was spun for 1 minute at fullspeed in an Eppendorf microfuge. The flow-through from collection tubewas discarded, and the column was placed back in the tube. Seven hundredand fifty μl of PE buffer was added, and the column spun for anotherminute at full speed. After discarding the flow-through from collectiontube, the column was spun for another minute at full speed to remove anyresidual PE buffer from the column. The column was transferred into aclean, microfuge tube, and 30 μl H₂O was added to the column andincubated for at least a minute at room temp. The column was spun forone minute at full speed. The PCR product/H₂O eluate in the microfugetube and was ready to be added to the sequencing reaction.

Example 4 Detection of European-like PRRSV

In this example, viral DNAs were amplified using primers that amplifyEuropean-like PRRSV, European PRRSV, and North American PRRSV. Theamplified region included the deletion that is present in European-likePRRSV.

The viral RNA of Lelystad was obtained from supernatants of infectedMA-104 cells, the viral RNA of VR-2332 was obtained from supernatants ofinfected MA-104 cells, and the viral RNA from European-like PRRSV wasobtained from supernatants of infected primary porcine alveolarmacrophages, cDNA of viral RNA was prepared as described above inExample 3.

The viral cDNAs were amplified using the primers Euro1671/:5′-GCCTGTCCTAACGCCAAGTAC (SEQ ID NO:16) and/Euro3165-rc:5′-CATGTCCACCCTATCCCACAT (SEQ ID NO:17). The amplification conditionsare listed in Table 2.

TABLE 2 General PCR Conditions (for 50 uL reaction) Stock ComponentConcentration Final Cone MgCl₂ 25 mM   5 mM Buffer II¹ 10 X   1 XForward Primer 15 uM  0.3 uM Reverse Primer 15 uM  0.3 uM Taq Polymerase 5 U/ul 0.25 U/ul ¹Buffer II, manufactured by Perkin Elmer.

RESULTS

Amplification of viral DNA from Lelystad, VR-2332, and European-likeresulted in amplification products that migrated at the predictedmolecular weights. As expected, the product of amplifying theEuropean-like DNA migrated at about 1.5 kilobases, approximately 51 basepairs less than Lelystad.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described for variations obvious to one skilled in theart will be included within the invention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Sequence Listing Free Text SEQ ID NO: 1 Portion of nucleotide sequenceof a porcine repro- ductive and respiratory syndrome virus SEQ ID NOs:2–10 Polypeptides predicted from open reading frames of SEQ ID NO: 1.SEQ ID NO: 11 Nucleotides 1,981 to 2,820 of the Lelystad virus SEQ IDNOs: 12–17 Primer SEQ ID NO: 18 Oligonucleotide SEQ ID NOs: 19–21 Primer

1. A method for detecting the presence of a European-like PRRSV,comprising: contacting a viral polynucleotide with a first primer and asecond primer under conditions suitable to form a detectableamplification product, wherein the first primer comprises a nucleotidesequence that is complementary to a portion of SEQ ID NO:1 thatcomprises nucleotides 2268 and 2269 of SEQ ID NO:1 or the complementthereof; and detecting an amplification product, wherein the detectionindicates that the viral polynucleotide is a European-like PRRSV.
 2. Themethod of claim 1 wherein the first primer comprises a nucleotidesequence selected from the group consisting of 5′ATCGGGAATGCTCAGTCCCCTT(SEQ ID NO:12), and 5′-AAGGGGACTGAGCATTCCCG (SEQ ID NO:14).
 3. A methodfor detecting the presence of a European-like PRRSV in a porcine subjectcomprising: contacting a biological sample obtained from a porcinesubject with a first primer and a second primer and incubating underconditions suitable to form a detectable amplification product, whereinthe first primer comprises a nucleotide sequence that is complementaryto a portion of SEQ ID NO:1 that comprises nucleotides 2268 and 2269 ofSEQ ID NO: 1 or the complement thereof; and detecting an amplificationproduct, wherein the detection indicates that the porcine subject has aEuropean-like PRRSV.
 4. The method of claim 3 wherein the first primercomprises a nucleotide sequence selected from the group consisting of5′ATCGGGAATGCTCAGTCCCCTT (SEQ ID NO:12), and 5′-AAGGGGACTGAGCATTCCCG(SEQ ID NO:14).
 5. The method of claim 3 wherein the biological samplecomprises lung tissue.
 6. A kit for use in detecting PRRSV in a porcinesubject, the kit comprising the first primer and a second primer ofclaim 1 or 3 for use in amplification of a portion of a PRRSV andinstructions for using the primer pair.